Vasoactive intestinal polypeptide pharmaceuticals

ABSTRACT

Pharmaceutical compositions relating to vasoactive intestinal polypeptides and methods for the treatment of metabolic disorders, including diabetes, insulin resistance, metabolic acidosis and obesity are presented. Methods of using the vasoactive intestinal polypeptide compositions are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.11/245,499 filed Oct. 7, 2005, which claims benefit of provisionalapplication 60/617,500 filed Oct. 8, 2004. This application is acontinuation-in-part of International Application No. PCT/US2005/036235filed Oct. 7, 2005.

FIELD OF THE INVENTION

The invention relates to polypeptide analogs and their synthesis anduses. More particularly, the invention relates to synthetic polypeptideanalogs related to vasoactive intestinal polypeptide, and pharmaceuticalcompositions thereof.

BACKGROUND

When food is present in the alimentary canal, cells in the gut secrete ahormonal signal (an “incretin”), which sensitizes the pancreas to thepresence of glucose and results in a potentiated glucose-dependentinsulin secretory response. Such a synergistic response to provideglucose-dependent insulin release (Kieffer T J and Habener, J R (1999)Endocr. Rev. 20, 876-913) is seen for the incretin signals,Glucagon-like Peptide 1 (GLP1) and Glucose-dependent InsulinotropicPeptide (GIP). These incretin signals typically exhibit short durationof action in the body, with GLP1 exhibiting at ½ of approximately 1-2minutes (Knudsen, L B 2004, J. Med. Chem. 47, 4128-34). GLP1 and GIP arecleaved by an amino peptidase, dipeptidyl peptidase IV (DPPIV) and thus,the naturally occurring native hormone is not generally used inmedicinal formulations. A peptide found in the saliva of the GilaMonster (exendin 4, Exenatide, Byetta; Amylin Pharmaceuticals, SanDiego, Calif.) was shown to bind to the GLP1 receptor and exhibit potentagonistic activity, thereby imparting a desirable glucose-dependentinsulin secretory response (Nielsen L L, Young, A A, Parkes, D G (2004)Regul. Peptides, 117, 77-88). Exenatide and analogs of GLP1 have beenadministered to patients in need of treatment for type 2 diabetes.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP) is aneuromodulatory peptide which stimulates PAC1, VPAC1, and VPAC2receptors, and is emitted from nerve endings in the pancreas. Receptorsof this general class reside in multiple tissues in the body, includingin the pancreas (Vaudry D, et al. (2000). Pharmacol Rev 52: 269-324).Administration (infusion) of PACAP to human volunteers or to rodentscauses potentiated glucose-dependent insulin secretion, but also resultsin hyperglycemia (Filipsson K, Tornoe K, Holst J and Ahren B (1997). JClin Endocrinol Metab 82: 3093-8). In contrast, Vasoactive IntestinalPolypeptide (VIP) activates only the VPAC1 and VPAC2 receptors. In thepancreas, stimulation of the VPAC2 receptors has been shown to provide apotentiated, glucose-dependent insulin release in response to elevatedblood glucose levels similar to that of GLP1 or exenatide (Tsutsumi, M.,et al. (2002) Diabetes 51, 1453-60). Thus such a stimulus (from PACAP orVPAC agonistic analogs) could be synergistic or alternative toincretin-like signals in stimulating glucose-dependent insulin release,since a similar profile of potentiated insulin secretion results fromactivation of a second class of receptor. Such an effect would bebeneficial in the treatment of metabolic disorders, including Type 2Diabetes Mellitus (T2DM), metabolic acidosis, insulin resistance andobesity. However, the lack of blood glucose lowering by PACAP in vivo isthought to be related to its ability to cause gluconeogenesis in theliver and release of glucagon. These activities, as well as several sideeffects, are believed to be caused by activation of PAC1 and VPAC1receptors. In addition, the naturally occurring native sequence of PACAPand its analogs also are typically short-lived in the body.

Although the reptile GLP1 like molecules (exendin-4, heliodermin,heliospectrin) are longer acting than the mammalian incretins, syntheticexendin-4 (Byetta) remains a relatively short acting peptide (t½ 2 hr inman) and there is a medical need for longer-acting peptides that canmodulate glucose-dependent insulin secretion.

SUMMARY OF THE INVENTION

The invention provides synthetic polypeptide analogs of PACAP andVasoactive Intestinal Polypeptide (VIP), and salts thereof, in which theC-terminus comprises amino acid residues that form an amphipathicα-helix, said residues selected from hydrophilic amino acids (Haa) andlipophilic amino acids (Laa) ordered in the sequence:Haa(Laa Laa Haa Haa)_(n)Laa.

wherein n−1-5. In an embodiment, n=1 or 2.

Modifications introduced in the present polypeptide analogs of PACAP andVIP facilitate increased duration of action of therapeutics whichactivates the PACAP and VIP family of receptors, preferably the VPAC2receptor. Without being bound to any particular theory, it is believedthat an increase in duration of action may be due to the ability of theamphipathic helix in the C-terminal region to interact with thephospholipids of the cell membranes in the body and thereby have a“depoting” effect. Thus, the present peptide analogs are bound to cellmembranes and then slowly re-released to the plasma to impart its effectdistally. In contrast, if a peptide such as PACAP, VIP or GLP1 is freein the plasma it is rapidly acted upon by proteases or cleared byglomerular filtration (Nestor J J Jr. Improved Duration of Action ofPeptide Drugs. In Peptide-based Drug Design: Taylor M D, Amidon G L,Eds.; American Chemical Society Washington D.C., 1995: 449-471).

Therefore, one aspect of the invention provides analogs to PACAP, VIP,and the physiologically active truncated analogs and homologs of same,or salts thereof, in which the C-terminus comprises amino acid residuesthat form an amphipathic α-helix, the sequence of said residues selectedfrom the native amino acids or selected unnatural amino acids having theability to stabilize said α-helix.

Also provided are pharmaceutical compositions for the delivery of aneffective glucose-dependant insulin releasing amount of a polypeptideanalog of PACAP, VIP, and the physiologically active truncated analogsand homologs of same, or a salt thereof, in which the C-terminuscomprises amino acid residues that form an amphipathic α-helix, saidresidues selected from hydrophilic amino acids (Haa) and lipophilicamino acids (Laa) ordered in the sequence:Haa(Laa Laa Haa Haa)_(n)Laa;

wherein n=1-5.

The invention further provides methods for treating mammalian conditionscharacterized by high blood glucose, which methods compriseadministering to a mammal in need thereof an effective glucose-dependantinsulin releasing amount of a polypeptide analog of PACAP, VIP, and thephysiologically active truncated analogs and homologs of same, or a saltthereof, in which the C-terminus comprises amino acid that form anamphipathic α-helix, said residues selected from hydrophilic amino acids(Haa) and lipophilic amino acids (Laa) ordered in the followingsequence:Haa(Laa Laa Haa Haa)_(n)Laa;

wherein n=1-5.

The invention further provides methods for treating mammalian conditionsaffected by VPAC receptor activation, which methods compriseadministering to a mammal in need thereof an effective glucose-dependantinsulin releasing amount of a polypeptide analog of PACAP, VIP, and thephysiologically active truncated analogs and homologs of same, or a saltthereof, in which the C-terminus comprises amino acid that form anamphipathic α-helix, said residues selected from hydrophilic amino acids(Haa) and lipophilic amino acids (Laa) ordered in the followingsequence:Haa(Laa Laa Haa Haa)_(n)Laa;

wherein n=1-5.

The invention also includes processes for the solid phase synthesis ofpolypeptide analogs of PACAP, VIP, and the physiologically activetruncated analogs and homologs of same, or a salt thereof, in which theC-terminus comprises amino acid residues that form an amphipathicα-helix, said residues selected from hydrophilic amino acids (Haa), andlipophilic amino acids (Laa) ordered in the following sequence:Haa(Laa Laa Haa Haa)_(n)Laa;

wherein n=1-5.

Processes presented herein for preparing polypeptide analogs comprisesequentially coupling protected amino acids on a suitable resin support,removing the side chain and Nα-protecting groups, and cleaving thepolypeptide from the resin.

The invention also provides DNA sequences, vectors, and plasmids for therecombinant synthesis of polypeptide analogs of PACAP, VIP, and thephysiologically active truncated analogs and homologs of same, or a saltthereof, in which the C-terminus comprises amino acid residues that forman amphipathic α-helix, said residues selected from hydrophilic aminoacids (Haa) and lipophilic amino acids (Laa) ordered in the sequence:Haa(Laa Laa Haa Haa)_(n)Laa;

wherein n=1-5.

In addition, the invention provides pharmaceutical compositions andmethods for the prevention and treatment of a variety of metabolicdisorders, including diabetes, insulin resistance, hyperglycemia,metabolic acidosis and obesity, which are manifested by elevated bloodglucose levels, comprising an effective amount of the polypeptide(s) ofthe invention, or salt thereof, and a pharmaceutically acceptablecarrier. In other aspects of the invention, therapeutically effectiveamounts of metabolic disorder compounds, including insulin, insulinanalogs, incretin, incretin analogs, glucagon-like peptide,glucagon-like peptide analogs, glucose dependent insulinotropic peptideanalogs, exendin, exendin analogs, sulfonylureas, biguanides,α-glucosidase inhibitors, thiazolidinediones, peroxisome proliferatoractivated receptor (PPAR) agonists, PPAR antagonists and PPAR partialagonists may be administered in combination with the polypeptides of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, and 1E are lists of exemplary polypeptide analogsaccording to the invention. Standard nomenclature using single letterabbreviations for amino acids are used. The letter “X” refers to apolyethylene glycol chain having C₁₀-C₃₀₀₀ chain. Preferred polyethylenechains may be linear or branched and will have a molecular weight above20 kiloDalton. The term “acyl” refers to a C₂-C₃₀ acyl chain. This chainmay comprise a linear aliphatic chain, a branched aliphatic chain, anaralkyl chain, or an aryl chain containing an acyl moiety. The letter“Z” refers to lysine having a long acyl chain at the epsilon position.The term “hex” refers to hexanoyl. The term “pen” refers to pentanoyl.The terms “lau” refers to lauroyl. The term “myr” refers to myristoyl.The term “ste” refers to stearoyl. The term “pr” refers to propionyl.Arachidoyl refers to a linear C20 fatty acid substituent.

FIG. 2 lists other polypeptide and polypeptide analogs.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions

The one- and three-letter abbreviations for the various commonnucleotide bases and amino acids are as recommended in Pure Appl. Chem.31, 639-645 (1972) and 40, 277-290 (1974) and comply with 37 CFR.sctn.1.822 (55 FR 18245, May 1, 1990). The abbreviations representL-amino acids unless otherwise designated as D- or DL. Certain aminoacids, both natural and non-natural, are achiral, e.g., glycine. Allpeptide sequences are presented with the N-terminal amino acid on theleft and the C-terminal amino acid on the right.

“Hydrophilic amino acid (Haa)” refers to an amino acid having at leastone hydrophilic functional group in addition to those required forpeptide bond formation, such as arginine, asparagine, aspartic acid,glutamic acid, glutamine, histidine, lysine, serine, threonine, andtheir homologs.

“Lipophilic amino acid (Laa)” refers to an uncharged, aliphatic oraromatic amino acid, such as isoleucine, leucine, methionine,phenylalanine, tryptophan, tyrosine, valine, and their homologs.

In the invention, alanine is classified as “amphiphilic” i.e., capableof acting as either hydrophilic or lipophilic.

“Homolog of PACAP or VIP” refers to a polypeptide comprising amino acidsin a sequence that is substantially similar to the native sequence ofPACAP or VIP, such as at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94,95, 96, 97, 98, or 99% amino acid sequence identity. Homologs presentedherein may comprise amino acid substitutions, deletions, and/orinsertions relative to the native sequence of PACAP or VIP. Exemplaryhomologs comprise a span of at least 5, 10, 15, 20, 25, 30, or 35contiguous amino acids that is identical or substantially similar to thenative sequence of PACAP or VIP.

“Analogs of PACAP or VIP” refers to a polypeptide comprising: (i) PACAP,VIP, and/or homologs of PACAP or VIP; and (ii) at least onefunctionality not present in naturally occurring native PACAP and/orVIP. For example, analogs can optionally comprise a functionality withinthe sidechain of an amino acid or at the amino or carboxyl terminal ofthe polypeptide. Exemplary functionalities include alkyl-, aryl-, acyl-,keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl,alkynyl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho,phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester,thioacid, hydroxylamine, amino group, or the like or any combinationthereof. Other exemplary functionalities that can be introduced include,but are not limited to, amino acids comprising a photoactivatablecross-linker, spin-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids thatcovalently or noncovalently interact with other molecules, photocagedand/or photoisomerizable amino acids, amino acids comprising biotin or abiotin analogue, glycosylated amino acids such as a sugar substitutedserine, other carbohydrate modified amino acids, keto containing aminoacids, amino acids comprising polyethylene glycol or polyether, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, e.g., polyethers or long chain hydrocarbons, e.g.,greater than about 5 or greater than about 10 carbons, carbon-linkedsugar-containing amino acids, redox-active amino acids, amino thioacidcontaining amino acids, and amino acids comprising one or more toxicmoiety.

Analogs presented herein may comprise non-natural amino acids based onnatural amino acids, such as tyrosine analogs include para-substitutedtyrosines, ortho-substituted tyrosines, and meta substituted tyrosines,wherein the substituted tyrosine comprises an acetyl group, a benzoylgroup, an amino group, a hydrazine, an hydroxyamine, a thiol group, acarboxy group, an isopropyl group, a methyl group, a C₆-C₂₀ straightchain or branched hydrocarbon, a saturated or unsaturated hydrocarbon,an O-methyl group, a polyether group, a nitro group, or the like.Glutamine analogs include, but are not limited to, α-hydroxyderivatives, β-substituted derivatives, cyclic derivatives, and amidesubstituted glutamine derivatives. Examples of phenylalanine analogsinclude, but are not limited to, meta-substituted phenylalanines,wherein the substituent comprises a hydroxy group, a methoxy group, amethyl group, an allyl group, an acetyl group, or the like. Specificexamples include, but are not limited to, O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAc.beta.-serine, an L-Dopa, a fluorinatedphenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine,a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, anL-phosphoserine, a phosphonoserine, a phosphonotyrosine, ap-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine,and an isopropyl-L-phenylalanine, and the like.

Generally, analogs are optionally designed or selected to modify thebiological properties of the polypeptide, such as to modulate toxicity,biodistribution, solubility, stability, e.g., thermal, hydrolytic,oxidative, resistance to enzymatic degradation, and the like, facilityof purification and processing, structural properties, spectroscopicproperties, chemical and/or photochemical properties, catalyticactivity, redox potential, half-life, ability to react with othermolecules, e.g., covalently or noncovalently, and the like.

“Physiologically active truncated homolog or analog of PACAP or VIP”refers to a polypeptide having a sequence comprising less than the fullcomplement of amino acids found in PACAP or VIP which, however, elicitsa similar physiological response. Representative truncated homologsand/or analogs presented herein comprise at least 5, 10, 15, 20, 25, 30,or 35 contiguous amino acids found in the native sequence of PACAP orVIP. The truncated PACAP or VIP need not be fully homologous with PACAPor VIP to elicit a similar physiological response. PACAP or VIP arepreferred, but not exclusive, representatives of this group.

“PEG” refers to polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes attached to the peptide or protein through a linkerfunctional group. Preferred forms of PEG have a molecular weight ofgreater than 10,000 Daltons and most preferred forms have a molecularweight of 20,000 Daltons or greater. They may be linear or branchedmolecules. Polyethyleneglycol chains are functionalized to allow theirconjugation to reactive groups on the polypeptide or protein chain.Typical functional groups allow reaction with amino, carboxy orsulfhydryl groups on the peptide through the corresponding carboxy,amino or maleimido groups (and the like) on the polyethylene glycolchain. In an embodiment, PEG comprises a C₁₀-C₃₀₀₀ chain. In anotherembodiment, PEG has a molecular weight above 40,000 Daltons. PEG as aprotein modification is well known in the art and its use is described,for example, in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192; and 4,179,337.

“Amphipathic α-helix” refers to the secondary structure exhibited bycertain polypeptides in which the amino acids assume an α-helicalconfiguration having opposing polar and nonpolar faces oriented alongthe long axis of the helix. The possibility of α-helical structure inthe polypeptide of interest may be explored to some extent by theconstruction of a “Schiffer-Edmundson wheel” (M. Schiffer and A. B.Edmundson, Biophys. J. 7, 121 (1967), incorporated herein by reference),of the appropriate pitch and noting the segregation of the hydrophilicand lipophilic residues on opposite faces of the cylinder circumscribingthe helix. Alternatively, empirical evidence, such as circular dichroismor x-ray diffraction data, may be available indicating the presence ofan α-helical region in a given polypeptide. An ideal α-helix has 3.6amino acid residues per turn with adjacent side chains separated by 100°of arc.

Another aspect of protein structure relevant to the invention, and inparticular those compounds of the strature corresponding to Formula II,is the use of a polyproline type II helix (Stapley B J and Creamer T P(1999) Protein Sci 8: 587-95) to facilitate the development of theamphiphilic alpha helix described above. Polyproline helicesincreasingly are recognized as being an important element in proteinstructure and an important aspect of that helix is its amphiphiliccharacter. Here we make use of such a polyproline type II helix tofacilitate that formation of the amphiphilic alpha helix described aboveto yield potent VPAC2 ligands. A prominent feature of polyprolinehelices is the very strong preference for Pro residues within the helixand specific amino acids as capping residues at the N-terminus. Someexamples of favored capping residues are Gln, Ser, Gly, Asp, Ala, Arg,Lys, Glu (Rucker A L, et al. (2003) Proteins 53: 68-75).

Another aspect of the polyproline helix is the resistance to proteolysisthat it affords. A number of naturally occurring peptides and proteinshave polyproline regions or Pro residues at their C-terminus, where theymay also prevent proteolytic digestion. Examples that bind to the GLP1receptor are Exendin-4, heliodermin, heliospectin.

Unless stated otherwise, standard nomenclature using single letterabbreviations for amino acids are used. The letter “X” refers to apolyethylene glycol chain having C₁₀-C₃₀₀₀ chain. Preferred polyethylenechains may be linear or branched and will have a molecular weight above20 kiloDalton. The term “acyl” refers to a C₂-C₃₀ acyl chain. This chainmay comprise a linear aliphatic chain, a branched aliphatic chain, anaralkyl chain, or an aryl chain containing an acyl moiety. The letter“Z” refers to lysine having a long acyl chain at the epsilon position.The term “hex” refers to hexanoyl. The term “pen” refers to pentanoyl.The terms “lau” refers to lauroyl. The term “myr” refers to myristoyl.The term “ste” refers to stearoyl. The term “pr” refers to propionyl.Arachidoyl refers to a linear C20 fatty acid substituent.

Although it may be apparent to an ordinary person skilled in the art, aPEG entity itself does not have a functional group to be attached to atarget molecular, such as a peptide. Therefore, to create PEGattachment, a PEG entity need to be functionalized first, then afunctionalized attachment is used to attach the PEG entity to a targetmolecule, such as a peptide. Site-specific pegylation can be achievedthrough Cys substitution on a peptide molecular. The target peptide canbe synthesized by solid phase synthesis or recombinant means describedherein. The invention discloses the combination concept of usingacylation on a Lys residue and specific pegylation on at least one Cysresidue. Certain Lys residues in disclosed peptide sequences can besubstituted to Cys for site-specific pegylation.

Polypeptides

In an embodiment, polypeptides presented herein comprise truncatedportions of PACAP and/or VIP having at least 5, 10, 15, 20, 25, 30, or35 contiguous amino acids of the native sequence of PACAP or VIP. Inanother embodiment, the present polypeptides share at least 50, 60, 70,80, 85, 90, 95, or 99% amino acid sequence identity to the nativesequence of PACAP or VIP. In yet another embodiment, the presentpolypeptides comprise a span of at least 5, 10, 15, 20, 25, 30, or 35contiguous amino acids of PACAP and/or VIP having at least 50, 60, 70,80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% amino acidsequence identity to the native sequence of PACAP or VIP.

Polypeptides presented herein optionally comprise modifications,functionalities, and/or amino acid substitutions which modulate VPAC2selectivity. Exemplary modifications, functionalities, and/orsubstitutions include, but are not limited to, C-terminal cationicextensions and/or mutations reported in Gourlet et al. (Peptides 18,403-8; Xia M, et al. (1997) and J. Pharmacol. Exp. Ther. 281: 629-33(1997); the contents of both of which are incorporated herein byreference).

Modifications at the amino or carboxyl terminus may optionally beintroduced into the present polypeptides. For example, the presentpolypeptides, such as analogs of VIP, can be acylated on the N-terminusby long chain fatty acids to yield polypeptides exhibiting low efficacy,partial agonist and antagonist activity (Gourlet et al., Eur. J.Pharmacol. 354: 105-111 (1998), the contents of which is incorporatedherein by reference). Modification of the peptides of this inventionwith longer chain fatty acids at the N-terminus, similarly will yieldantagonists with a prolonged duration of action (Moreno D, et al. (2000)Peptides 21: 1543-9). Other modifications to the N-terminus, such asdeletions or incorporation of D-amino acids such as D-Phe also givepotent and long acting antagonists when substituted into the compoundsof Formulas 1-3. Such antagonists also have commercial utility and arewithin the scope of this invention.

Other exemplary modifications of the present polypeptides, such asanalogs of VIP, include acylation with hexanoic acid to yieldpolypeptides that exhibit increased selectivity towards VPAC2 (Langer etal., Peptides 25: 275-8 (2004), the contents of which is incorporatedherein by reference). Thus the length and positioning of such acylationcan alter efficacy, and could result in loss of efficacy (antagonistic)or agonistic analogs. Contrary to this unpredictability, polypeptidespresented herein have been designed and tested to obtain desiredefficacy and activity.

Modifications may optionally be introduced within the side chain of atleast one amino acid within the present polypeptides to increaseduration of action and/or potency. For example, the present polypeptidescan optionally comprise at least one amino acid acylated to afunctionality in the side chain (i.e., R group). Representativemodifications include fatty acid acylation, directly or through linkers,of reactive side chains (such as Lys) at various positions within thepolypeptide. Similar modifications have been reported in Kurtzhals etal. where acylation of insulin on LysB²⁹ resulted in insulin detemir(Kurtzhals et al., (1995) Biochem J. 312, 725-31: and Kurtzhals, P.,(2004) Int. J. Obesity 28: Suppl 2, S23-8). Similarly, acylation withlong chain fatty acids through linkers (preferably Glu) has resulted inpotent and long-acting analogs of GLP1 (Knudsen L B, et al. (2000). J.Med. Chem. 43, 1664-69), but the acylation can result in loss ofactivity or potent agonists, depending on the length and positioning ofthe acyl chain(s). Contrary to the unpredictable effects with theintroduction of long chain fatty acids, polypeptides presented hereinhave been designed to incorporate an optimal number, length andpositioning of the acyl chains so as to obtain desired activity. Suchlinkage is demonstrated here for direct acylation to Lys, but linkagethrough other linkers, such as Glu (Knudsen, L B, et al. (2000)), isalso within the scope of the present invention.

Another type of modification that can optionally be introduced into thepresent polypeptides (e.g. within the polypeptide chain or at either theN- or C-terminal) to extend duration of action is PEGylation orincorporation of long-chain polyethylene glycol polymers (PEG).Introduction of PEG or long chain polymers of PEG increases theeffective molecular weight of the present polypeptides to prevent rapidfiltration into the urine. Any Lys residue in any peptide analogsequence may be conjugated to PEG directly or through a linker to yielda potent and long acting analog. Such linker can be a Glu residue or anacyl residue containing a thiol functional group for linkage to theappropriately modified PEG chain. An alternative method for introducinga PEG chain is to first introduce a Cys residue at the C-terminus or atsolvent exposed residues such as replacements for Arg or Lys residues.This Cys residue is then site-specifically attached to a PEG chaincontaining, for example, a maleimide function. Methods for incorporatingPEG or long chain polymers of PEG are well known in the art anddescribed, for example, in Greenwald et al., Adv. Drug Del. Rev. 55:217-250 (2003), the contents of which is incorporated herein byreference.

A more recently reported alternative approach for incorporating PEG orPEG polymers through incorporation of non-natural amino acids can beperformed with the present polypeptides. This approach utilizes anevolved tRNA/tRNA synthetase pair and is coded in the expression plasmidby the amber suppressor codon (Deiters, A, et al. (2004). Bio-org. Med.Chem. Lett. 14, 5743-5). For example, p-azidophenylalanine can beincorporated into the present polypeptides and then reacted with a PEGpolymer having an acetylene moiety in the presence of a reducing agentand copper ions to facilitate an organic reaction known as “Huisgen[3+2] cycloaddition”.

Amphipathic Helix

Polypeptides of the present invention comprise amphipathic α-helixcorresponding to the formula:Haa(Laa Laa Haa Haa)_(n)Laawherein n, Haa, and Laa are selected from the group of hydrophilic aminoacids and the Laa's are selected from the group of lipophilic aminoacids, as defined above. Without being bound to any particular theory,it is believed that the amphipathic helix in the C-terminal regionimparts an increase in duration of action of the present polypeptides byinteracting with the phospholipids of the cell membranes in the body andthereby have a “depoting” effect. Polypeptides of the present inventioncomprise a peptide region that is an amphipathic alpha helix, not merelyan alpha helix. Without wishing to be bound by any particular theory,the amphipathic alpha helix is believed to facilitate increasedinteraction with cell membranes and assist in proper placement ofC-terminal fatty acyl chain modifications for membrane interaction.

Studies by Eisenberg et al. have combined a hydrophobicity scale withthe helical wheel to quantify the concept of amphipathic helices (Nature299: 371-374 (1982) and Proc. Nat. Acad. Sci. USA 81: 140-144 (1984);the disclosures of which are hereby incorporated by reference). The meanhydrophobic moment is defined as the vector sum of the hydrophobicitiesof the component amino acids making up the helix. The followinghydrophobicities for the amino acids are those reported by Eisenberg etal. as the “consensus” scale: Ile 0.73; Phe 0.61; Val 0.54; Leu 0.53;Trp 0.37; Met 0.26 Ala 0.25; Gly 0.16; Cys 0.04; Tyr 0.02; Pro −0.07;Thr −0.18; Ser −0.26; His −0.40; Glu −0.62; Asn −0.64; Gln −0.69; Asp−0.72; Lys −1.10; Arg −1.76.

The hydrophobic moment, μH, for an ideal α-helix having 3.6 residues perturn (or a 100° arc (=360°/3.6) between side chains), may be calculatedfrom:μH=[(ΣH_(N) sine δ(N−1)²+(ΣH_(N) COS δ(N−1))₂]^(1/2),where H_(N) is the hydrophobicity value of the N^(th) amino acid and thesums are taken over the N amino acids in the sequence with periodicityδ=100°. The hydrophobic moment may be expressed as the mean hydrophobicmoment per residue by dividing μH by N to obtain <μH>. A value of <μH>at 100°+−0.20° of about 0.20 or greater is suggestive of amphipathichelix formation.

A study by Cornett et al. has further extended the study of amphiphathicα-helices by introducing the “amphipathic index” as a predictor ofamphipathicity (J. Mol. Biol., 195: 659-685 (1987); the disclosure ofwhich is hereby incorporated by reference). They concluded thatapproximately half of all known α-helices are amphipathic, and that thedominant frequency is 97.5° rather than 100°, with the number ofresidues per turn being closer to 3.7 than 3.6. The basic approach ofEisenberg, et al. is sufficient to classify a given sequence asamphipathic, particularly when one is designing a sequence ab initio toform an amphipathic α-helix.

A substitute amphipathic α-helical amino acid sequence may lack homologywith the sequence of a given segment of a naturally occurringpolypeptide but elicits a similar secondary structure, i.e. an α-helixhaving opposing polar and nonpolar faces, in the physiologicalenvironment. Replacement of the naturally occurring amino acid sequencewith an alternative sequence may beneficially affect the physiologicalactivity, stability, or other properties of the altered parentpolypeptide. Exemplary reports describing the design and selection ofsuch sequences is provided in J. L. Krstenansky, et al., FEBS Letters242: 2, 409-413 (1989), and J. P. Segrest, et al. Proteins: Structure,Function, and Genetics 8: 103-117 (1990) among others.

Polypeptides of the present invention comprise amphipathic α-helixcorresponding to the formula:Haa(Laa Laa Haa Haa)_(n)Laawherein the Haa's are selected from the group of hydrophilic amino acidsand the Laa's are selected from the group of lipophilic amino acids, asdefined above. Assuming an idealized α-helix in an embodiment where n=2,residues 1, 4, 5, 8, and 9 are distributed along one face (A) of thehelix within about a 140° arc of each other, while residues 2, 3, 6, 7,and 10 occupy an opposing 140° arc on the other face (B) of the helix.In an embodiment, all the residues on one face are of the same polaritywhile all those on the other face are of the opposite polarity, i.e., ifface A is all hydrophilic, face B is all lipophilic and vice versa. Theskilled artisan will recognize that while the helices of the inventionare described byHaa(Laa Laa Haa Haa)_(n)Laa,the reverse sequence,Laa(Haa Haa Laa Laa)_(n)Haawill also meet the residue distribution criteria and is an equivalentdescriptor of the helices of the invention.

Alanine may be substituted for either hydrophilic or lipophilic aminoacids, since Ala can reside readily on either face of an amphipathicα-helix, although Ala-10 does not form an amphipathic α-helix.Generally, proline, cysteine, and tyrosine are not used; however, theirpresence and other random errors in the sequence may be tolerated, e.g.a hydrophilic residue on the lipophilic face, as long as the remainingamino acids in the segment substantially conform to the hydrophilicface—lipophilic face division. A convenient method for determining if asequence is sufficiently amphipathic to be a sequence of this inventionis to calculate the mean hydrophobic moment, as defined above. If thepeak mean moment per residue at 100°+−20° exceeds about 0.20, then thesequence will form an amphipathic helix and is a sequence of theinvention.

In applying this concept to PACAP and VIP, it is hypothesized thateither or both regions (N-terminal or C-terminal), preferably theC-terminal, may exhibit α-helical secondary structure and could bereplaced with a non-homologous sequence having similar structuraltendencies, without loss of biological activity or induction ofimmunoreaction.

Pharmaceutical Formulations

Polypeptides of the present invention may be administered in any amountto impart beneficial therapeutic effect. In a preferred embodiment,compounds of the present invention are useful in the treatment ofelevated blood glucose levels, hyperglycemia, diabetes, including Type 2Diabetes Mellitus, insulin resistance, metabolic acidosis and obesity.In an embodiment, compounds presented herein impart beneficial activityin the modulation of insulin and/or glucose levels. In an embodiment,the present polypeptides are administered to a patient at concentrationshigher or lower than that of other forms of treatment which modulateinsulin and/or glucose secretion. In yet another embodiment, the presentpolypeptides are administered with other compounds to produce asynergistic therapeutic effect. For example, polypeptides of theinvention may be administered in conjunction with exendin or exendinanalogs.

EXAMPLES Example 1 Synthetic Analogs

Some of the exemplary synthetic polypeptide analogs illustrated in FIG.1 are derived from VPAC2 sel UldB (see FIG. 1). Other exemplarysynthetic polypeptide analogs illustrated in FIG. 1 are truncatedhomologs of VIP (see FIG. 1).

In one aspect, the present polypeptide analogs of the physiologicallyactive truncated homologs of VIP, such as those shown in FIG. 1 as TP 1to TP 6. Analogs TP 1 to TP 6 have a long acyl residue comprisingC12-C24, preferably C16-C24. Analogs TP 7 to TP 12 shown in FIG. 1 havean acyl residue on the N-terminus comprising C2-C16, preferably C6.Analogs SQNM 10-12 (corresponding to SEQ ID NO: 40-42) shown in FIG. 2do not contain acylation at either the C or N-termini.

Other representative polypeptide analogs presented herein have aminoacid sequences corresponding to general formula (I):

Formula (I) (SEQ ID NO: 81)Acyl-His-Ser-Asp-aa₁-aa₂-Phe-Thr-aa₃-aa₄-Tyr-aa₅-Arg-aa₆-Baa₁-Baa₂-Baa₃-aa₇-Ala-aa₈-Baa₄-Baa₅-Tyr-Leu-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-Haa-(Laa-Laa-Haa-Haa)_(n)-Laa-Lys(ε-long acyl)-Xwherein:

-   -   n=1-5;    -   each Haa is independently a hydrophilic amino acid;    -   each Laa is independently a lipophilic amino acid;    -   acyl is a C₂₋₁₆ acyl chain;    -   long acyl is a C₁₂₋₃₀ acyl chain;    -   X is OH or NHR1 where R1 is H or lower alkyl, haloalkyl, or        Cys(PEG)-X, or PEG;    -   PEG is a functionalized polyethylene glycol chain of C₁₀-C₃₀₀₀        chain;    -   aa₁ is Gly or Ala;    -   aa₂ is Val, Lie, or Leu;    -   aa₃ is Asp, Arg, Gln, or Glu;    -   aa₄ is Ser, Asn, Gln, Asp or Glu;    -   aa₅ is Ser or Thr;    -   aa₆ is Leu or Tyr;    -   Baa₁ is Arg or Leu;    -   Baa₂ is Lys, Leu, or Arg;    -   Baa₃ is Gln, Lys or Ala;    -   aa₇ is Met, Leu, Val or Ala    -   aa₈ is Ala or Val;    -   Baa₄ is Lys, Arg or Gln;    -   Baa₅ is Lys, Arg or Gln;    -   aa₉ is Asn, Gln, Ala or Glu;    -   aa₁₀ is Trp, Ala, or Ser;    -   aa₁₁ is Ile, Val or Trp;    -   aa₁₂ is Leu, Lys, Arg or Gln;    -   aa₁₃ is Lys, Arg, Asn, Gln, or Gly;    -   aa₁₄ is Ala, Gly, Gln, Lys or Arg;    -   aa₁₅ is Lys, Arg, Leu, Ala or absent; and    -   aa₁₆ is Lys, Arg, Leu, Ala or absent.        provided that if aa₁₅ or aa₁₆ is absent, the next amino acid        present downstream is the next amino acid in the peptide agonist        sequence. In a preferred embodiment, acyl is a C₂₋₈ acyl chain        and long acyl is a C₁₂₋₃₀ acyl chain.

Other representative polypeptide analogs presented herein have aminoacid sequences corresponding to general formula (II):

Formula (II) (SEQ ID NO: 82)Acyl-His-Ser-Asp-aa₁-aa₂-Phe-Thr-aa₃-aa₄-Tyr-aa₅-Arg-aa₆-Baa₁-Baa₂-Baa₃-aa₇-Ala-aa₈-Baa₄-Baa₅-Tyr-Leu-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-Haa-(Laa-Laa-Haa-Haa)_(n)-Laa-aa₁₇-Pro-Pro-Pro-Lys(ε-long acyl)-Xwherein:

-   -   n=1-5;    -   each Haa is independently a hydrophilic amino acid;    -   each Laa is independently a lipophilic amino acid;    -   acyl is a C₂₋₁₆ acyl chain;    -   long acyl is a C₁₂₋₃₀ acyl chain;    -   X is OH or NHR1 where R1 is H or lower alkyl, haloalkyl, or        Cys(PEG)-X, or PEG;    -   PEG is a functionalized polyethylene glycol chain of C₁₀-C₃₀₀₀        chain;    -   aa₁ is Gly or Ala;    -   aa₂ is Val, Ile, or Leu;    -   aa₃ is Asp, Arg, Gln, or Glu;    -   aa₄ is Ser, Asn, Gln, Asp or Glu;    -   aa₅ is Ser or Thr;    -   aa₆ is Leu or Tyr;    -   Baa₁ is Arg or Leu;    -   Baa₂ is Lys, Leu, or Arg;    -   Baa₃ is Gln, Lys or Ala;    -   aa₇ is Met, Leu, Val or Ala    -   aa₈ is Ala or Val;    -   Baa₄ is Lys, Arg or Gln;    -   Baa₅ is Lys, Arg or Gln;    -   aa₉ is Asn, Gln, Ala or Glu;    -   aa₁₀ is Trp, Ala, or Ser;    -   aa₁₁ is Ile, Val or Trp;    -   aa₁₂ is Leu, Lys, Arg or Gln;    -   aa₁₃ is Lys, Arg, Asn, Gln, or Gly;    -   aa₁₄ is Ala, Gly, Gln, Lys or Arg;    -   aa₁₅ is Lys, Arg, Leu, Ala or absent;    -   aa₁₆ is Lys, Arg, Leu, Ala or absent; and    -   aa₁₇ is Gln, Ser, Gly, Asp, Ala, Arg, Lys, Glu, Pro, Asn, Leu,        or absent.        provided that if aa₁₅, aa₁₆ or aa₁₇ is absent, the next amino        acid present downstream is the next amino acid in the peptide        agonist sequence. In a preferred embodiment, acyl is a C₂₋₈ acyl        chain and long acyl is a C₁₂₋₃₀ acyl chain.

Other representative polypeptide analogs presented herein have aminoacid sequences corresponding to general formula (III):

Formula (III) Acyl-His-Ser-Asp-aa₁-aa₂-Phe-Thr-aa₃-aa₄-Tyr-aa₅-Arg-aa₆-Baa₁-Baa₂-Baa₃-aa₇-Ala-aa₈-Baa₄-Baa₅-Tyr-Leu-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-Haa-(Laa-Laa-Haa-Haa)_(n)-Laa-aa₁₇-Lys(ε-long acyl)-PEGwherein:

-   -   n=1-5;    -   each Haa is independently a hydrophilic amino acid;    -   each Laa is independently a lipophilic amino acid;    -   acyl is a C₂₋₁₆ acyl chain;    -   long acyl is a C₁₂₋₃₀ acyl chain;    -   PEG is a functionalized polyethylene glycol chain of C₁₀-C₃₀₀₀        chain;    -   aa₁ is Gly or Ala;    -   aa₂ is Val, Ile, or Leu;    -   aa₃ is Asp, Arg, Gln, or Glu;    -   aa₄ is Ser, Asn, Gln, Asp or Glu;    -   aa₅ is Ser or Thr;    -   aa₆ is Leu or Tyr;    -   Baa₁ is Arg or Leu;    -   Baa₂ is Lys, Leu, or Arg;    -   Baa₃ is Gln, Lys or Ala;    -   aa₇ is Met, Leu, Val or Ala;    -   aa₈ is Ala or Val;    -   Baa₄ is Lys, Arg or Gln;    -   Baa₅ is Lys, Arg or Gln;    -   aa₉ is Asn, Gln, Ala or Glu;    -   aa₁₀ is Trp, Ala, or Ser;    -   aa₁₁ is Ile, Val or Trp;    -   aa₁₂ is Leu, Lys, Arg or Gln;    -   aa₁₃ is Lys, Arg, Asn, Gln, or Gly;    -   aa₁₄ is Ala, Gly, Gln, Lys or Arg;    -   aa₁₅ is Lys, Arg, Leu, Ala or absent;    -   aa₁₆ is Lys, Arg, Leu, Ala or absent; and    -   aa₁₇ is Gln, Ser, Gly, Asp, Ala, Arg, Lys, Glu, Pro, Asn, Leu,        or absent.        provided that if aa₁₅, aa₁₆ or aa₁₇ is absent, the next amino        acid present downstream is the next amino acid in the peptide        agonist sequence. In a preferred embodiment, acyl is a C₂₋₈ acyl        chain and long acyl is a C₁₂₋₃₀ acyl chain.

The skilled artisan will appreciate that numerous permutations of thepolypeptide analogs may be synthesized which will possess the desirableattributes of those described herein provided that an amino acidsequence having a mean hydrophobic moment per residue at 100°+−20°greater than about 0.20 is inserted at positions in the C-terminalregion.

Example 2 Additional Analogs

In some embodiments of the invention, representative polypeptide analogspresented herein have the following amino acid sequences:

Formula (IV) Acyl-aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-Thr-aa₈-aa₉-aa₁₀-Thr-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-Ala-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-aa₂₇-aa₂₈-aa₂₉-aa₃₀-aa₃₁-aa₃₂-aa₃₃-aa₃₄-aa₃₅-aa₃₆-aa₃₇-aa₃₈-aa₃₉-aa₄₀wherein:

-   -   aa₁ is: any naturally occurring amino acid, dH, or is absent;    -   aa₂ is: any naturally occurring amino acid, dA, dS, or Aib;    -   aa₃ is: Asp or Glu;    -   aa₄ is: any naturally occurring amino acid, dA, Aib, or NMeA;    -   aa₅ is: any naturally occurring amino acid, dV, or Aib;    -   aa₆ is: any naturally occurring amino acid;    -   aa₈ is: Asp, Glu, Ala, Lys, Leu, Arg, or Tyr;    -   aa₉ is: Asn, Gln, Asp, or Glu;    -   aa₁₀ is: any naturally occurring aromatic amino acid, or Tyr        (OMe);    -   aa₁₂ is: hR, Orn, Lys (isopropyl), Aib, Cit or any naturally        occurring amino acid except Pro;    -   aa₁₃ is: Aib, or any naturally occurring amino acid except Pro;    -   aa₁₄ is: hR, Orn, Lys (isopropyl), Aib, Cit, or any naturally        occurring amino acid except Pro;    -   aa₁₅ is: hR, Orn, Lys (isopropyl), Aib, K (Ac), Cit, or any        naturally occurring amino acid except Pro;    -   aa₁₆ is: hR, Orn, Lys (isopropyl), Cit, or any naturally        occurring amino acid except Pro;    -   aa₁₇ is: Nle, Aib, or any naturally occurring amino acid except        Pro;    -   aa₁₉ is: any naturally occurring amino acid except Pro;    -   aa₂₀ is: hR, Orn, Lys (isopropyl), Aib, K(Ac), Cit, or any        naturally occurring amino acid except Pro;    -   aa₂₁ is: hR, Orn, Aib, K(Ac), Cit, or any naturally occurring        amino acid except Pro;    -   aa₂₂ is: Aib, Tyr (OMe), or any naturally occurring amino acid        except Pro;    -   aa₂₃ is: Aib, or any naturally occurring amino acid except Pro;    -   aa₂₄ is: any naturally occurring amino acid except Pro;    -   aa₂₅ is: Aib, or any naturally occurring amino acid except Pro;    -   aa₂₆ is: any naturally occurring amino acid except Pro;    -   aa₂₇ is: hR, Lys (isopropyl), Orn, dK, or any naturally        occurring amino acid except Pro;    -   aa₂₈ is: any naturally occurring amino acid, Aib, hR, Cit, Gm,        dK, or is absent;    -   aa₂₉ is: any naturally occurring amino acid, hR, Orn, Cit, Aib        or is absent;    -   aa₃₀ is: any naturally occurring amino acid, hR, Orn, Cit, Aib        or is absent; and    -   aa₃₁ to aa₄₀ are any naturally occurring amino acid or are        absent;        provided that if aa₁, aa₂₈, aa₂₉, aa₃₀, aa₃₁, aa₃₂, aa₃₃, aa₃₄,        aa₃₅, aa₃₆, aa₃₇, aa₃₈, aa₃₉, or aa₄₀ is absent, the next amino        acid present downstream is the next amino acid in the peptide        agonist sequence;        and a C-terminal sequence selected from the group consisting of:    -   (a) -Haa-(Laa-Laa-Haa-Haa)_(n)-Laa-Lys(ε-long acyl)-X;    -   (b) -Haa-(Laa-Laa-Haa-Haa)_(n)-Laa-aa₁₇-Pro-Pro-Pro-Lys(ε-long        acyl)-X; (SEQ ID NO: 84)    -   (c) -Haa-(Laa-Laa-Haa-Haa)_(n)-Laa-Lys(s-long acyl)-PEG; and    -   (d) a polyproline type II helix;        wherein:    -   n is an integer number from 1 to 3;    -   each Haa is independently a hydrophilic amino acid;    -   each Laa is independently a lipophilic amino acid;    -   acyl is a C₂₋₁₆ acyl chain;    -   long acyl is a C₁₂₋₃₀ acyl chain;    -   X is OH or NHR1 where R1 is H or lower alkyl, haloalkyl, or PEG.

In some embodiments of the invention, representative polypeptide analogspresented herein have the following amino acid sequences:

Formula (V) Acyl-aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-Thr-aa₈-aa₉-aa₁₀-Thr-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-Ala-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-aa₂₇-aa₂₈-aa₂₉-aa₃₀-aa₃₁-aa₃₂-aa₃₃-aa₃₄-aa₃₅-aa₃₆-aa₃₇-aa₃₈-aa₃₉-aa₄₀wherein:

-   -   aa₁ is: His, dH, or is absent;    -   aa₂ is: dA, Ser, Val, Gly, Thr, Leu, dS, Pro, or Aib;    -   aa₃ is: Asp or Glu;    -   aa₄ is: Ala, Ile, Tyr, Phe, Val, Thr, Leu, Trp, Gly, dA, Aib, or        NMeA;    -   aa₅ is: Val, Leu, Phe, Ile, Thr, Tm, Tyr, dV, Aib, or NMeV;    -   aa₆ is: Phe, Ile, Leu, Thr, Val, Trp, or Tyr;    -   aa₈ is: Asp, Glu, Ala, Lys, Leu, Arg, or Tyr;    -   aa₉ is: Asn, Gln, Asp, or Glu;    -   aa₁₀ is: Tyr, Trp, or Tyr(OMe);    -   aa₁₂ is: Arg, Lys, Glu, hR. Orn, Lys (isopropyl), Aib, Cit, or        Ala;    -   aa₁₃ is: Leu, Phe, Glu, Ala, or Aib;    -   aa₁₄ is: Arg, Leu, Lys, Ala, hR, Orn, Lys (isopropyl), Phe, Gln,        Aib, or Cit;    -   aa₁₅ is: Lys, Ala, Arg, Glu, Leu, hR. Orn, Lys (isopropyl), Phe,        Gln, Aib, K(Ac), or Cit;    -   aa₁₆ is: Gln, Lys, Glu, Ala, hR. Orn, Lys (isopropyl), or Cit;    -   aa₁₇ is: Val, Ala, Leu, Ile, Met, Nle, Lys, or Aib;    -   aa₁₉ is: Val, Ala, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,        Pro, Gln, Arg, Ser, Thr, Trp, Tyr, Cys, or Asp;    -   aa₂₀ is: Lys, Gln, hR, Arg, Ser, His, Orn, Lys (isopropyl), Ala,        Aib, Trp, Thr, Leu, Ile, Phe, Tyr, Val, K(Ac), or Cit;    -   aa₂₁ is: Lys, His, Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR , K(Ac)        or Cit;    -   aa₂₂ is: Tyr, Trp, Phe, Thr, Leu, Ile, Val, Tyr(OMe), Ala, or        Aib;    -   aa₂₃ is: Leu, Phe, Ile, Ala, Trp, Thr, Val, or Aib;    -   aa₂₄ is: Gln, Glu, or Asn;    -   aa₂₅ is: Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, Tyr,        Aib, or Glu;    -   aa₂₆ is: Ile, Leu, Thr, Val, Trp, Tyr, Phe or Aib    -   aa₂₇ is: Lys, hR. Arg, Gln, Ala, Asp, Glu, Phe, Gly, His, Ile,        Met, Asn, Pro, Ser, Thr, Val, Trp, Tyr, Lys (isopropyl), Cys,        Leu, Orn, or dK;    -   aa₂₈ is: Asn, Asp, Gln, Lys, Arg, Aib, Orn, hR, Cit, Pro, dK, or        is absent;    -   aa₂₉ is: Lys, Ser, Arg, Asn, hR, Ala, Asp, Glu, Phe, Gly, His,        Ile, Leu, Met, Pro, Gln, Thr, Val, Trp, Tyr, Cys, Orn, Cit, Aib        or is absent;    -   aa₃₀ is: Arg, Lys, Ile, Ala, Asp, Glu, Phe, Gly, His, Leu, Met,        Asn, Pro, Gln, Ser, Thr, Val, Trp, Tyr, Cys, hR. Cit, Aib, Orn,        or is absent;    -   aa₃₁ is: Tyr, His, Phe, Thr, Cys, or is absent;    -   aa₃₂ is: Ser, Cys, or is absent;    -   aa₃₃ is: Trp or is absent;    -   aa₃₄ is: Cys or is absent;    -   aa₃₅ is: Glu or is absent;    -   aa₃₆ is: Pro or is absent;    -   aa₃₇ is: Gly or is absent;    -   aa₃₈ is: Trp or is absent;    -   aa₃₉ is: Cys or is absent; and    -   aa₄₀ is: Arg or is absent;        provided that if aa₁, aa₂₈, aa₂₉, aa₃₀, aa₃₁, aa₃₂, aa₃₃, aa₃₄,        aa₃₅, aa₃₆, aa₃₇, aa₃₈, aa₃₉, or aa₄₀ is absent, the next amino        acid present downstream is the next amino acid in the peptide        agonist sequence;        and a C-terminal sequence selected from the group consisting of:    -   (a) -Haa-(Laa-Laa-Haa-Haa)_(n)-Laa-Lys(ε-long acyl)-X;    -   (b) -Haa-(Laa-Laa-Haa-Haa)_(n)-Laa-aa₁₇-Pro-Pro-Pro-Lys(ε-long        acyl)-X; (SEQ ID NO: 85)    -   (c) -Haa-(Laa-Laa-Haa-Haa)_(n)-Laa-Lys(s-long acyl)-PEG; and    -   (d) a polyproline type II helix;        wherein:    -   n is an integer number from 1 to 3;    -   each Haa is independently a hydrophilic amino acid;    -   each Laa is independently a lipophilic amino acid;    -   acyl is a C₂₋₁₆ acyl chain;    -   long acyl is a C₁₂₋₃₀ acyl chain;    -   X is OH or NHR1 where R1 is H or lower alkyl, haloalkyl, or PEG.

Example 3 General Method for Synthesizing Polypeptides

The polypeptides of the invention may be synthesized by methods such asthose set forth in J. M. Stewart and J. D. Young, Solid Phase PeptideSynthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984) and J.Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, NewYork, (1973) for solid phase synthesis and E. Schroder and K. Lubke, ThePeptides, Vol. 1, Academic Press, New York, (1965) for solutionsynthesis and Houben-Weyl, Synthesis of Peptides and Peptidomimetics.4th ed. Vol E22; M. Goodman, A. Felix, L. Moroder, C. Toniolo, Eds.,Thieme: New York, 2004 for general synthesis techniques. Microwaveassisted peptide synthesis is an attractive method and will be aparticularly effective method of synthesis for the peptides of theinvention (Erdelyi M, et al. (2002) Synthesis 1592-6). The disclosuresof the foregoing treatises are incorporated by reference herein.

In general, these methods involve the sequential addition of protectedamino acids to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid and any reactive side chain groupare protected. This protected amino acid is then either attached to aninert solid support, or utilized in solution, and the next amino acid inthe sequence, also suitably protected, is added under conditionsamenable to formation of the amide linkage. After all the desired aminoacids have been linked in the proper sequence, protecting groups and anysolid support are removed to afford the crude polypeptide. Thepolypeptide is desalted and purified, preferably chromatographically, toyield the final product.

A preferred method of preparing the analogs of the physiologicallyactive truncated polypeptides, having fewer than about forty aminoacids, involves solid phase peptide synthesis. In this method theα-amino (Nα) functions and any reactive side chains are protected byacid- or base-sensitive groups. The protecting group should be stable tothe conditions of peptide linkage formation, while being readilyremovable without affecting the extant polypeptide chain. Suitableα-amino protecting groups include, but are not limited tot-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz),o-chlorobenzyloxycarbonyl, biphenylisopropyloxycarbonyl,t-amyloxycarbonyl (Amoc), isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxy-carbonyl, o-nitrophenylsulfenyl,2-cyano-t-butoxycarbonyl, 9-fluorenyl-methoxycarbonyl (Fmoc) and thelike, preferably t-butoxycarbonyl (Boc). Suitable side chain protectinggroups include, but are not limited to: acetyl, benzyl (Bzl),benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, t-butyl,t-butyldimethylsilyl, 2-chlorobenzyl (Cl-z), 2,6-dichlorobenzyl,cyclohexyl, cyclopentyl, isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl(Tos), trimethylsilyl, and trityl.

In solid phase synthesis, the C-terminal amino acid is first attached toa suitable resin support. Suitable resin supports are those materialswhich are inert to the reagents and reaction conditions of the stepwisecondensation and deprotection reactions, as well as being insoluble inthe media used. Examples of commercially available resins includestyrene/divinylbenzene resins modified with a reactive group, e.g.,chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylatedco-poly-(styrene-divinylbenzene), and the like. Benzylated,hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred. Whenthe C-terminus of the compound is an amide, a preferred resin isp-methylbenzhydrylamino-co-poly(styrene-divinyl-benzene) resin.

Attachment to the PAM resin may be accomplished by reaction of the Nαprotected amino acid, preferably the Boc-amino acid, as its ammonium,cesium, triethylammonium, 1,5-diazabicyclo-[5.4.0]undec-5-ene,tetramethylammonium, or similar salt in ethanol, acetonitrile,N,N-dimethylformamide (DMF), and the like, preferably the cesium salt inDMF, with the resin at an elevated temperature, for example betweenabout 40° and 60° C., preferably about 50° C., for from about 12 to 72hours, preferably about 48 hours.

The Nα-Boc-amino acid may be attached to the benzhydrylamine resin bymeans of, for example, an N,N′-diisopropylcarbodiimide(DIC)/1-hydroxybenzotriazole (HOBt) mediated coupling for from about 2to about 24 hours, preferably about 2 hours at a temperature of betweenabout 10° and 50° C., preferably 25° C. in a solvent such asdichloromethane or dimethylformamide, preferably dichloromethane.

The successive coupling of protected amino acids may be carried out bymethods well known in the art, typically in an automated peptidesynthesizer. Following neutralization with triethylamine or similarbase, each protected amino acid is preferably introduced inapproximately 1.5 to 2.5 fold molar excess and the coupling carried outin an inert, nonaqueous, polar solvent such as dichloromethane, DMF, ormixtures thereof, preferably in dichloromethane at ambient temperature.Representative coupling agents are N,N′-dicyclohexylcarbodiimide (DCC),N,N′-diisopropyl-carbodiimide (DIC) or other carbodiimide, either aloneor in the presence of 1-hydroxybenzotriazole (HOBt), O-acyl ureas,benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate(PyBop), N-hydroxysuccinimide, other N-hydroxyimides, or oximes.Alternatively, protected amino acid active esters (e.g. p-nitrophenyl,pentafluorophenyl and the like) or symmetrical anhydrides may be used.

At the end of the solid phase synthesis the fully protected peptide isremoved from the resin. When the linkage to the resin support is of thebenzyl ester type, cleavage may be effected by means of aminolysis withan alkylamine or fluoroalkylamine for peptides with an alkylamideC-terminus, or by aminolysis with, for example, ammonia/methanol orammonia/ethanol for peptides with an unsubstituted amide C-terminus, ata temperature between about −10° and 50° C., preferably about 25° C.,for between about 12 and 24 hours, preferably about 18 hours. Peptideswith a hydroxy C-terminus may be cleaved by HF or other strongly acidicdeprotection regimen or by saponification. Alternatively, the peptidemay be removed from the resin by transesterification, e.g., withmethanol, followed by aminolysis or saponification. The protectedpeptide may be purified by silica gel chromatography.

The side chain protecting groups may be removed from the peptide bytreating the aminolysis product with, for example, anhydrous liquidhydrogen fluoride in the presence of anisole or other carbonium ionscavenger, treatment with hydrogen fluoride/pyridine complex, treatmentwith tris(trifluoroacetyl)boron and trifluoroacetic acid, by reductionwith hydrogen and palladium on carbon or polyvinylpyrrolidone, or byreduction with sodium in liquid ammonia, preferably with liquid hydrogenfluoride and anisole at a temperature between about −10° and +10° C.,preferably at about 0° C., for between about 15 minutes and 2 hours,preferably about 1.5 hours.

For peptides on the benzhydrylamine resin, the resin cleavage anddeprotection steps may be combined in a single step utilizing liquidhydrogen fluoride and anisole as described above.

The solution may be desalted (e.g. with BioRad AG-3® anion exchangeresin) and the peptide purified by a sequence of chromatographic stepsemploying any or all of the following types: ion exchange on a weaklybasic resin in the acetate form; hydrophobic adsorption chromatographyon underivatized co-poly(styrene-divinylbenzene), e.g. Amberlite® XAD;silica gel adsorption chromatography; ion exchange chromatography oncarboxymethylcellulose; partition chromatography, e.g. on Sephadex®G-25; counter-current distribution; or high performance liquidchromatography (HPLC), especially reversed-phase HPLC on octyl- oroctadecylsilylsilica (ODS) bonded phase column packing.

Thus, another aspect of the present invention relates to processes forpreparing polypeptides and pharmaceutically acceptable salts thereof,which processes comprise sequentially condensing protected amino acidson a suitable resin support, removing the protecting groups and resinsupport, and purifying the product, to afford analogs of thephysiologically active truncated homologs and analogs of PACAP and VIP,preferably of PACAP and VIP in which the amino acids at the C-terminusform an amphipathic α-helical peptide sequence, as defined above.

Example 4 Exemplary Synthesis and Purification Protocol for aRepresentative Polypeptide Analog

Representative polypeptide analog corresponding to SEQ ID NO: 1 wasprepared using the synthetic and purification methods described below.

(SEQ ID NO: 1) Pentanoyl-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Val-Ala-Ala-Lys-Lys-Tyr-Leu-Asn-Trp-Ile-Lys-Lys-Ala-Lys-Arg-Glu-Leu-Leu- Glu-Lys-Leu-Lys(epsilonstearoyl)-NH₂

Generally, the peptide was synthesized on Fmoc-Rink-Amide-PEG resin viaFmoc chemistry. Protecting groups used for amino acid side chainfunctional groups are: t-Butyl group for Glu, Tyr, Thr, Asp and Ser; Bocgroup for Lys and Trp; Pbf group for Arg; Trt group for Asn and His.N-alpha Fmoc protected amino acids were purchased from EMD Biosciences(San Diego, Calif.). Reagents for coupling and cleavage were purchasedfrom Aldrich (St. Louis, Mo.). Solvents were purchased from FisherScientific (Fairlawn, N.J.).

Generally, the synthetic protocol involved assembly of the peptide chainon resin by repetitive removal of the Fmoc protecting group and couplingof protected amino acid. For the synthesis, Dde-Lys(Fmoc)-OH was coupledonto the Rink Amide resin first. The Fmoc protecting group was thenremoved by 20% piperidine in DMF. Stearic acid was coupled onto the sidechain of Lys using HBTU, HOBt and NMM. The Dde group was removed by 2%hydrazine in DMF and the next Fmoc protected amino acid was coupled.HBTU and HOBt were used as coupling reagent and NMM was used as base.After removal of last Fmoc protecting group, valeric acid (4equivalents) was coupled to the amino terminus with DIC (4 equivalents)and HOBt (4 equivalents). The peptide resin was treated with cocktail 1for cleavage and removal of the side chain protecting groups. Crudepeptide was precipitated from cold ether and collected by filtration.

Purification of crude peptide was achieved via RP-HPLC using 20 mm×250mm column from Waters (Milford, Mass.). Peptide was purified using TFABuffer. A linear gradient of 35% to 55% acetonitrile in 60 minutes wasused. Pooled fractions were lyophilized. The peptide identity wasverified by mass spectrometry analysis and amino acid analysis. Thepeptide purity was determined by analytical HPLC column (C 18 column,4.6×250 mm, manufactured by Supelco (St. Louis, Mo.)) chromatography.

The above procedure can be summarized in the following step wiseprotocol:

-   -   Step 1. Resin swelling: Fmoc-Rink-Amide-PEG resin was swelled in        DCM for 30 minutes (10 ml/g resin)    -   Step 2. Deprotection:        -   a. 20% piperidine/DMF solution (10 ml/g resin) was added to            the resin;        -   b. Solution stirred for 30 minutes (timing was started when            all the resin was free floating in the reaction vessel); and        -   c. Solution was drained.    -   Step 3. Washing: Resin was washed with DMF (10 ml/g resin) five        times. The ninhydrin test was performed and appeared positive.    -   Step 4. Coupling:        -   a. Fmoc-AA-OH (3 equivalents calculated relative to resin            loading) and HOBt (3 equivalents relative to resin loading)            was weighed into a plastic bottle.        -   b. Solids were dissolved with DMF (5 ml/g resin).        -   c. HBTU (3 equivalents relative to resin loading) was added            to the mixture, followed by the addition of NMM (6            equivalents relative to resin loading).        -   d. Mixture was added to the resin.        -   e. Mixture was bubbled (or stirred) gently for 10-60 minutes            until a negative ninhydrin test on a small sample of resin            was obtained.    -   Step 5. Washing: Resin was washed three times with DMF.    -   Step 6. Steps 2-5 were repeated until the peptide was assembled.    -   Step 7. N-terminal Fmoc Deprotection: Step 2 was repeated.    -   Step 8. Washing and Drying:        -   a. After the final coupling, resin was washed three times            with DMF, one time with MeOH, three times with DCM, and            three times with MeOH.        -   b. Resin was dried under vacuum (e.g., water aspirator) for            2 hours and high vacuum (oil pump) for a minimum of 12            hours.    -   Step 9. Cleavage:        -   a. Dry resin was placed in a plastic bottle and the cleavage            cocktail was added. The mixture was shaken at room            temperature for 2.5 hours.        -   b. The resin was removed by filtration under reduced            pressure. The resin was washed twice with TFA. Filtrates            were combined and an 8-10 fold volume of cold ether was            added to obtain a precipitate.        -   c. Crude peptides were isolated by filtration and then            washed twice with cold ether.

The following chemicals and solvents were used in the synthetic protocoldescribed above: NMM (N-Methylmorpholine); HBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumHexafluorophosphate); HOBt (1-Hydroxybenzotriazole); DMF(Dimethylformamide); DCM (Dichloromethane); Methanol; Diethylether;Piperidine; Tis (Triisopropylsilane); Thioanisole; Phenol; EDT(1,2-Ethanedithiol); Trifluoroacetic acid Cocktail 1:TFA/Thioanisole/Phenol/H₂O/EDT (87.5/5/2.5/2.5/2.5 v/v/); TFA buffer: A(0.1% TFA in water); and TFA buffer B (0.1% TFA in Acetonitrile).

Other representative polypeptide analogs were prepared in a mannersimilar to that described above. Listed below in TABLE 1 are chemicalproperties of exemplary polypeptide analogs of the invention.

TABLE 1 Properties of Exemplary Polypeptide Analogs Molecular WeightBased on Name of Amino Purity Based on RP- Electrospray Analog AcidSequence HPLC Chromatogram Mass Spectrometry TP-103 SEQ ID NO: 2 96.9%5267.2 a.m.u. TP-104 SEQ ID NO: 3 95.5% 4756.7 a.m.u. TP-105 SEQ ID NO:4 96.1% 5183.3 a.m.u. TP-106 SEQ ID NO: 5 95.2% 4784.8 a.m.u. TP-107 SEQID NO: 6 99.6% 4955.1 a.m.u. TP-108 SEQ ID NO: 7 91.5% 5172.4 a.m.u.

Example 5 Recombinant Synthesis of the Polypeptides

Alternatively, the polypeptides of the present invention may be preparedby cloning and expression of a gene encoding for the desiredpolypeptide. In this process, a plasmid containing the desired DNAsequence is prepared and inserted into an appropriate hostmicroorganism, typically a bacteria, such as E. coli, or a yeast, suchas Saccharomyces cerevisiae, inducing the host microorganism to producemultiple copies of the plasmid, and so of the cDNA encoding for thepolypeptide analogs of the invention.

First, a synthetic gene coding for the selected PACAP or VIP analog isdesigned with convenient restriction enzyme cleavage sites to facilitatesubsequent alterations. Polymerase chain reaction (PCR), as taught byMullis in U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporated herein byreference, may be used to amplify the sequence.

The amplified synthetic gene may be isolated and ligated to a suitableplasmid, such as a Trp LE plasmid, into which four copies of the genemay be inserted in tandem. Preparation of Trp LE plasmids is describedin U.S. Pat. No. 4,738,921 and European Patent Publication No. 0212532,incorporated herein by reference. Trp LE plasmids generally produce 8-10times more protein than Trp E plasmids. The multi-copy gene may then beexpressed in an appropriate host, such as E. coli or S. cerevisiae.

Trp LE 18 Prot (Ile3, Pro5) may be used as an expression vector in thepresent invention. Trp LE 18 Prot (Ile3, Pro5) contains the followingelements: a pBR322 fragment (EcoRI-BamHI) containing the ampicillinresistant gene and the plasmid origin of replication; an EcoRI-SacIIfragment containing the trp promoter and the trpE gene; an HIV protease(Ile3, Pro5) gene fragment (SacII-HindIII); a bGRF gene fragment(HindIII-BamHI); and a transcription terminator from E. coli rpoc gene.The HIV protease and bGRF gene fragments are not critical and may bereplaced with other coding sequences, if desired.

The expressed multimeric fusion proteins then accumulate intracellularlyinto stable inclusion bodies and may be separated by centrifugation fromthe rest of the cellular protein. VIP and PACAP related peptides do notdenature so purification is straightforward through a combined ionexchange concentration/purification protocol followed by “polishing” onpreparative reversed-phase high performance chromatography using aaqueous to aqueous-organic buffer gradient using 0.1% trifluoroaceticacid or 0.4M NH₄OAc (pH 4) as the pH modifier. The organic modifier usedmay be any of a number of water miscible solvents, for exampleacetonitrile, n-propanol, isopropanol, and the like, preferablyn-propanol. The isolated fusion protein is converted to the monomericPACAP or VIP analog by acylation with activated fatty acids and may bepurified by cation exchange and/or reverse phase HPLC. The preciseprotocol is dependent on the particular sequence being synthesized.Typically the free amino terminus is less reactive than a Lys sidechain, so differential acylation is straightforward. Alternatively, afragment of the final sequence may be prepared in this way withsubsequent condensation with a synthetically produced fragmentcontaining the N- or C-terminal modifications. Chemical or “native”conjugations may be used (Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.;Kent, S. B. Science 1994, 266, (5186), 776-9; Nilsson, B. L.; Soellner,M. B.; Raines, R. T. Annu Rev Biophys Biomol Struct 2005, 34, 91-118.).

Alternative methods of cloning, amplification, expression, andpurification will be apparent to the skilled artisan. Representativemethods are disclosed in Maniatis, et al., Molecular Cloning, aLaboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001),incorporated herein by reference.

Example 6 In Vitro Bioassay with Islet Cell Static Cultures

The following exemplary in vitro bioassay was conducted to evaluate theability of representative polypeptide analogs to modulate insulinsecretion.

Islet isolation. Rat islets were harvested (Sweet I R, et al. (2004)Biochem. Biophys. Res. Commun. 314, 976-983) from male Fisher ratsweighing about 250 g and which were anesthetized by intraperitonealinjection of sodium pentobarbital (35 mg/230 g rat). Generally, theislets were prepared by injecting collagenase (10 mL of 0.23 mg/mLLiberase, Roche Molecular Biochemicals, Indianapolis, Ind.) into thepancreatic duct of the partially dissected pancreas and surgicallyremoving it. All procedures were approved by the Institutional AnimalCare and Use Committee at the University of Washington.

The pancreata were placed into 15 mL conical tubes containing 5 mL of0.23 mg/mL Liberase and incubated at 37° C. for 30 min. The digestatewas then filtered through a 400-micrometer stainless steel screen,rinsed with Hanks' buffered salt solution, and purified in a gradientsolution of Optiprep (Nycomed, Oslo, Norway). Islets were cultured for18-24 h prior to performing the assay in RPMI Media 1640 supplementedwith 10% (v/v) heat inactivated fetal bovine serum (FBS),antibiotic-antimycotic (100 U/mL penicillin, 100 lg/mL streptomycin, and0.25 lg/mL amphotericin B), 2 mM glutamine (all from Gibco-BRL, GrandIsland, N.Y.), and 1 mM beta mercaptoethanol.

Bioassay. Islets were picked under a microscope and placed into 10 ml 3mM Krebs Ringer Buffer (KRB) solution for washing. Islets were incubatedin 3 mM glucose KRB for 60 min and then groups of 10 islets per wellwere placed into 200 μl media in a 96-well plate. The islets wereincubated for 120 min under control or treatment conditions, andsupernatants were collected. A typical set of conditions tested 3 mMglucose (resting control), 16 mM glucose (testing control), 16 mMglucose+10 nm GLP1, 16 mM glucose+10 nM Exendin-4, 16 mM glucose+50 nMtest peptide. The buffer conditions were KRB with 0.1% BSA, 20 mM HEPESand the assay was performed in quadruplicate. Supernatants wereevaluated for insulin content using a commercial insulin enzyme-linkedimmunosorbent (ELISA) assay per manufacturer's directions.

Results of Bioassay. TABLE 2 illustrates the insulin secretion obtainedin the above assay for analog TP-106, which exhibited maximal activityin this assay at a concentration of 200 nM. For comparison, Exendin 4was tested in this assay and showed maximal activity at 10 nM. TP-106 isa highly hydrophobic analog, designed to depot in the site of scinjection and therefore the effective concentration of TP-106 isexpected to be much lower than the nominal concentration (200 nM).

TABLE 2 Results of Islet Cell Static Culture Bioassay with TP-106Insulin secreted Standard (ng/100 islets/min) Deviation 3 mM glucose0.01 0.00 16 mM glucose 1.38 0.17 Exendin 4 + 16 mM glucose 4.82 0.20 50nM TP-106 + 16 mM glucose 2.72 0.60 200 nM TP-106 + 16 mM glucose 5.200.50 16 mM glucose + 16 mM glucose 1.58 0.05

The islet cell static culture assay described above was performed onadditional exemplary polypeptide analogs. TP-107 exhibited maximalactivity in this assay at a concentration of 100 nM. For comparison,Exendin 4 was tested in this assay and showed maximal activity at 10 nM.Presented peptides were designed to bind to serum albumin and thus, theconcentration of free peptide to impart insulin activity is expected tobe much lower and therefore the analog more potent than indicated inthis in vitro assay.

TABLE 3 Results of Islet Cell Static Culture Bioassay with TP-107 andTP-108 Average Insulin secreted Standard (ng/100 islets/min) Deviation 3mM glucose 0.14 0.00 16 mM glucose 3.65 0.80 10 nM Exendin 4 + 16 mMglucose 6.75 1.15 10 nM PACAP + 16 mM glucose 6.07 1.67 10 nM TP-107 +16 mM glucose 2.89 0.21 100 nM TP-107 + 16 mM glucose 6.10 1.55 1 uMTP-107 + 16 mM glucose 6.07 0.90 100 nM TP-108 + 16 mM glucose 4.10 1.211 uM TP-108 + 16 mM glucose 5.65 0.13

Example 7 In Vivo Bioassay

The following exemplary in vivo assay was conducted to evaluate theability of representative polypeptide analogs to modulate insulinsecretion.

Tested Study Groups. Naïve, 8 weeks old female db/db mice wereacclimated for one week, during which period animals were handledperiodically to allow them to be acclimated to experiment procedures.Study groups contained 6 mice per group and were administered with oneof the following by intraperitoneal injection:

(1) Vehicle control;

(2) Positive control (exendin-4 or other standard treatment);

(3) Polypeptide Analog at high dose; or

(4) Polypeptide Analog at low dose.

A small volume of blood was taken from a cut at the tip of tail forblood sampling. Blood glucose levels were determined on a commercial,hand-held glucose meter. On Day 1, animals were injected withpolypeptide analogs and controls in the morning. Blood samples weretaken and analyzed immediately before injection and at 2, 4, 8, 14, and24 hours after injection. Animals were allowed to feed, ad libitem,throughout the assay (Tsutsumi et al., Diabetes 51:1453-60 (2002)).

TABLE 4 lists a representative sampling of the data obtained from the invivo assay described above. As shown below, TP-106 exhibitedstatistically significant activity (e.g., reduced plasma glucose) at ahigh dose 2 hr after injection and maintains activity at 4 hrs postdosing.

TABLE 4 Results of In Vivo Assay with TP-103 and TP-106 Mean BloodGlucose Levels (mmol/L) 0 hr 2 hr 4 hr 8 hr 14 hr 24 hr Vehicle 23.921.9 18.3 27.3 22.5 23.5 s.d.* = 1.33 s.d. = 1.22 s.d. = 1.01 s.d. =1.52 s.d. = 1.25 s.d. = 1.31 TP-103 Low dose 22.9 20.5 17.6 26.4 24.621.4 s.d. = 1.27 s.d. = 1.14 s.d. = 0.98 s.d. = 1.47 s.d. = 1.37 s.d. =1.19 TP-103 High dose 20.7 17.3 16.9 23.4 23.7 25.0 s.d. = 1.15 s.d. =0.96 s.d. = 0.94 s.d. = 1.30 s.d. = 1.31 s.d. = 1.39 TP-106 Low dose23.9 20.5 16.1 24.0 28.2 23.2 s.d. = 1.33 s.d. = 1.14 s.d. = 0.89 s.d. =1.33 s.d. = 1.57 s.d. = 1.29 TP-106 High dose 21.8 13.4 14.7 25.1 26.321.2 s.d. = 1.21 s.d. = 0.75 s.d. = 0.82 s.d. = 1.39 s.d. = 1.46 s.d. =1.18 *s.d. = standard deviation

Example 8 Uses of the Invention

The polypeptides of the present invention are useful for the preventionand treatment of a variety of metabolic disorders. In particular, thecompounds of the present invention are indicated for the prophylaxis andtherapeutic treatment of elevated blood glucose levels, hyperglycemia,diabetes, including Type 2 Diabetes Mellitus, Maturity Onset Diabetes ofthe Young (MODY), Metabolic Syndrome, insulin resistance, metabolicacidosis and obesity.

The polypeptides of the present invention are useful for the preventionand treatment of a variety of inflammatory disorders, defined broadly.In particular the compounds of the present invention are indicated forthe prophylaxis and therapeutic treatment of asthma (Linden A, et al.(2003). Thorax 58: 217-21), cardioprotection during ischemia (Kalfin, etal. (1994). J Pharmacol Exp Ther 1268: 952-8; Das, et al. (1998). Ann NYAcad Sci 865: 297-308), primary pulmonary hypertension (Petkov V, et al.(2003). J Clin Invest 111: 1339-46), and the like.

In another embodiment, the polypeptides of the invention may beadministered in combination with other compounds useful in the treatmentof metabolic disorders. For example, the polypeptides of the inventionmay be administered with one or more of the following compounds used inthe treatment of metabolic disorders, including but not limited toinsulin, insulin analogs, incretin, incretin analogs, glucagon-likepeptide, glucagon-like peptide analogs, glucose dependent insulinotropicpeptide analogs, exendin, exendin analogs, sulfonylureas, biguanides,α-glucosidase inhibitors, thiazolidinediones, peroxisome proliferatoractivated receptor (PPAR, of which includes agents acting on the α, β,or γ subtypes of PPAR receptors and/or those agent acting on multiplesubtypes of the PPAR receptors) agonists, PPAR antagonists and PPARpartial agonists may be administered in combination with thepolypeptides of the present invention.

In other contemplated disease applications, the peptides of theinvention can be used advantageously in coordination withpharmaceuticals currently applied for that disease. Particularlybeneficial are combination drug formulations containing mixtures of theactive pharmaceutical ingredients with excipients. For example, inasthma, the VPAC2 agonists can used in combination with inhaledformulations containing bronchodilators, beta 2 adrenoceptor agonists,inhaled corticosteroids, anti-inflammatory steroids, leukotrienemodifiers, leukotriene receptor antagonists, chemokine modifiers,chemokine receptor antagonists, cromolyn, nedocromil, xanthines,anticholinergic agents, immune modulating agents, other knownanti-asthma medications, and the like. Additional agents in development(Corry D B and Kheradmand F (2006) J Allergy Clin Immunol 117 (2 Suppl):S461-47) may also be beneficial when used in combination with VPAC2agonists.

VPAC2 combination treatments may make use of currently appliedtherapeutics for treatment of pulmonary hypertension, as well. Thus aVPAC2 agonist may be utilized in combination with nitric oxide donors,prostacyclins, endothelin antagonists, adrenoceptor blockers,phosphodiesterases inhibitors, ion channel blockers and othervasodilators (Levy J H (2005) Tex Heart Inst J 32: 467-71; Haj R M, etal. (2006) Curr Opin Anesthesiol 19: 88-95).

Representative delivery regimens include oral, parenteral (includingsubcutaneous, intramuscular and intravenous injection), rectal, buccal(including sublingual), transdermal, inhalation and intranasal. Anattractive and widely used method for delivery of peptides entailssubcutaneous injection of a controlled release injectable formulation.Preferred administration routes for the application of the peptides ofthe invention are subcutaneous, intranasal and inhalationadministration.

The selection of the exact dose and composition and the most appropriatedelivery regimen will be influenced by, inter alia, the pharmacologicalproperties of the selected polypeptide, the nature and severity of thecondition being treated, and the physical condition and mental acuity ofthe recipient. Additionally, the route of administration will result indifferential amounts of absorbed material. Bioavailabilities foradministration of peptides through different routes are particularlyvariable, with amounts from less than 1% to near 100% being seen.Typically, bioavailability from routes other than intravenous injectionare 50% or less.

In general, the polypeptides of the invention, or salts thereof, areadministered in amounts between about 0.1 and 60 μg/kg body weight perday, preferably from about 0.1 to about 1 μg/kg body weight per day, bysubcutaneous injection. For a 50 kg human female subject, the daily doseof active ingredient is from about 5 to about 1000 μg, preferably fromabout 5 to about 500 μg by subcutaneous injection. Different doses willbe needed, depending on the route of administration and the applicablebioavailability observed. By inhalation, the daily dose is from 100 toabout 5,000 μg, twice daily. In other mammals, such as horses, dogs, andcattle, higher doses may be required. This dosage may be delivered in aconventional pharmaceutical composition by a single administration, bymultiple applications, or via controlled release, as needed to achievethe most effective results, preferably one or more times daily byinjection.

Pharmaceutically acceptable salts retain the desired biological activityof the parent polypeptide without toxic side effects. Examples of suchsalts are (a) acid addition salts formed with inorganic acids, forexample hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid and the like; and salts formed with organic acids suchas, for example, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acids, naphthalene disulfonicacids, polygalacturonic acid and the like; (b) base addition saltsformed with polyvalent metal cations such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and thelike; or with an organic cation formed from N,N′-dibenzylethylenediamineor ethylenediamine; or (c) combinations of (a) and (b), e.g., a zinctannate salt and the like.

A further aspect of the present invention relates to pharmaceuticalcompositions comprising as an active ingredient a polypeptide of thepresent invention, or pharmaceutically acceptable salt thereof, inadmixture with a pharmaceutically acceptable, non-toxic carrier. Asmentioned above, such compositions may be prepared for parenteral(subcutaneous, intramuscular or intravenous) administration,particularly in the form of liquid solutions or suspensions; for oral orbuccal administration, particularly in the form of tablets or capsules;for intranasal administration, particularly in the form of powders,nasal drops or aerosols; for inhalation, particularly in the form ofliquid solutions or dry powders with excipients, defined broadly; andfor rectal or transdermal administration.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985), incorporated herein by reference. Formulations for parenteraladministration may contain as excipients sterile water or saline,alkylene glycols such as propylene glycol, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenatednaphthalenes, serum albumin nanoparticles (as used in Abraxane™,American Pharmaceutical Partners, Inc. Schaumburg Ill.), and the like.For oral administration, the formulation can be enhanced by the additionof bile salts or acylcarnitines. Formulations for nasal administrationmay be solid and may contain excipients, for example, lactose ordextran, or may be aqueous or oily solutions for use in the form ofnasal drops or metered spray. For buccal administration typicalexcipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

When formulated for nasal administration, the absorption across thenasal mucous membrane may be enhanced by surfactant acids, such as forexample, glycocholic acid, cholic acid, taurocholic acid, ethocholicacid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid,glycodeoxycholic acid, cyclodextrins and the like in an amount in therange between about 0.2 and 15 weight percent, preferably between about0.5 and 4 weight percent, most preferably about 2 weight percent. Anadditional class of absorption enhancers exhibiting greater efficacywith decreased irritation is the class of alkyl maltosides, such astetradecylmaltoside (Arnold, J J, et al. (2004). J Pharm. Sci. 93,2205-13, and references therein, all of which are hereby incorporated byreference).

When formulated for delivery by inhalation, a number of formulationsoffer advantages. Adsorption of the active peptide to readily dispersedsolids such as diketopiperazines (for example Technosphere particles;Pfutzner A and Forst T (2005). Expert Opin Drug Deliv 2: 1097-106.) orsimilar structures gives a formulation which results in a rapid initialuptake of the therapeutic agent. Lyophylized powders, especially glassyparticles, containing the active peptide and an excipient are useful fordelivery to the lung with good bioavailability, for example, seeExubera® (inhaled insulin by Pfizer and Aventis Pharmaceuticals Inc.).Additional systems for delivery of polypeptides by inhalation (Mandal TK (2005) Am J Health Syst Pharm 62: 1359-64) are well known in the artand are incorporated into this invention.

Delivery of the compounds of the present invention to the subject overprolonged periods of time, for example, for periods of one week to oneyear, may be accomplished by a single administration of a controlledrelease system containing sufficient active ingredient for the desiredrelease period. Various controlled release systems, such as monolithicor reservoir-type microcapsules, depot implants, osmotic pumps,vesicles, micelles, liposomes, transdermal patches, iontophoreticdevices and alternative injectable dosage forms may be utilized for thispurpose. Localization at the site to which delivery of the activeingredient is desired is an additional feature of some controlledrelease devices, which may prove beneficial in the treatment of certaindisorders.

One form of controlled release formulation contains the polypeptide orits salt dispersed or encapsulated in a slowly degrading, non-toxic,non-antigenic polymer such as copoly(lactic/glycolic) acid, as describedin the pioneering work of Kent, Lewis, Sanders, and Tice, U.S. Pat. No.4,675,189, incorporated by reference herein. The compounds or,preferably, their relatively insoluble salts, may also be formulated incholesterol or other lipid matrix pellets, or silastomer matriximplants. Additional slow release, depot implant or injectableformulations will be apparent to the skilled artisan. See, for example,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinsoned., Marcel Dekker, Inc., New York, 1978, and R. W. Baker, ControlledRelease of Biologically Active Agents, John Wiley & Sons, New York,1987, incorporated by reference herein.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the examples and discussion given above are intended to illustratethe synthesis and testing of representative compounds of the invention,it will be understood that it is capable of further modifications andshould not be construed as limiting the scope of the appended claims.

1. A vasoactive intestinal polypeptide of:Acyl-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Gln-Ty-Thr-Arg-Leu-Leu-Leu-Lys-Val-Ala-Ala-Lys-Lys-Tyr-Leu-Gln-Trp-Ile-Lys-Lys-Ala-Lys-Arg-Glu-Leu-Leu-Glu-Lys-Leu-Lys(ε-long acyl)-X SEQ ID NO: 65 wherein: Acyl is a C₂₋₁₆ acyl chain orbenzoyl; long acyl is a C₆₋₃₀ acyl chain; X is OH, NHR1, Cys(PEG), orPEG; R1 is H, lower alkyl, or haloalkyl; PEG is a functionalizedpolyethylene glycol chain of C₁₀-C₃₀₀.
 2. The polypeptide of claim 1,wherein acyl is a C₄-C₉ acyl chain; long acyl is a C₆-C₂₀ acyl chain;and PEG is a polyethylene glycol chain of C₁₀₀-C₃₀₀ chain.
 3. Thepolypeptide of claim 1, wherein Acyl is benzoyl.
 4. The polypeptide ofclaim 1, wherein long acyl is arachidyl and X is OH.
 5. A pharmaceuticalcomposition comprising an effective amount of the polypeptide of claim1, or acceptable salt thereof, and at least one pharmaceuticallyacceptable carrier or excipient.
 6. The pharmaceutical composition ofclaim 5, further comprising an effective amount of at least one compoundselected from the group consisting of insulin, insulin analogs,incretin, incretin analogs, glucagon-like peptide, glucagon-like peptideanalogs, glucose dependent insulinotropic peptide analogs, exendin,exendin analogs, sulfonylureas, biguanides, a-glucosidase inhibitors,thiazolidinediones, peroxisome proliferator activated receptor (PPAR)agonists, PPAR antagonists and PPAR partial agonists.
 7. A vasoactiveintestinal polypeptide of:Benzoyl-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Gln-Tyr-Thr-Arg-Leu-Leu-Leu-Lys-Vat-Ala-Ala-Lys-Lys-Tyr-Leu-Gln-Trp-Ile-Lys-Lys-Ala-Lys-Arg-Glu-Leu-Leu-Glu-Lys-Leu-Lys(ε-arachidoyl) SEQ ID NO:65.
 8. A pharmaceutical composition comprisingan effective amount of the polypeptide of claim 7, or acceptable saltthereof, and at least one pharmaceutically acceptable carrier orexcipient.
 9. The pharmaceutical composition of claim 8, furthercomprising an effective amount of at least one compound selected fromthe group consisting of insulin, insulin analogs, incretin, incretinanalogs, glucagon-like peptide, glucagon-like peptide analogs, glucosedependent insulinotropic peptide analogs, exendin, exendin analogs,sulfonylureas, biguanides, a-glucosidase inhibitors, thiazolidinediones,peroxisome proliferator activated receptor (PPAR) agonists, PPARantagonists and PPAR partial agonists.